Project Summary Atrial fibrillation (AF) is associated with significant morbidity and increased mortality. Despite recent advances in catheter-based treatments, antiarrhythmic drugs (AADs) are still commonly used to treat AF. However, response to membrane-active drugs is highly variable, in part, because of the failure to target therapy to the underlying mechanisms. Although genetic approaches have provided important insights into the underlying mechanisms of AF, the translation of these discoveries to the bedside care of patients has been limited due to the challenges of adequately recapitulating human AF in cellular models. The ability to derive patient-specific atrial cardiomyocytes (CMs) from human induced pluripotent stem cells (iPSC)-CMs holds great promise for modeling AF-linked mutations and developing cellular models of AF that are genetically-matched to specific patients. However, atrial iPSC-CMs have not been used to elucidate the underlying cellular mechanisms of AF- linked mutations and model heritable AF. Specific Aim 1 will create the UIC Multi-Ethnic Atrial HiPSC-CM Biorepository with the goal of generating atrial iPSC-CMs from familial AF kindreds to model AF-linked mutations. Our pilot data shows that atrial iPSC-CMs recapitulated the electrophysiologic (EP) phenotype of an AF-linked SCN5A mutation, and served as a platform for targeting the underlying cellular mechanism of the gain-of-function variant. Nonetheless, enhancing the maturity of iPSC-CMs remains important as modeling mature CMs will not only provide additional insights into the underlying cellular mechanisms of AF but also identify molecular signaling pathways important for atrial development. Specific Aim 2 will test the hypothesis that the EP and structural maturity of atrial iPSC-CMs can be significantly enhanced by precise microenvironmental engineering of in-vivo relevant cell-cell, cell-extracellular matrix, and cell-soluble factor interactions, such that these cells allow for optimal modeling of AF. We will also assess the role of cardiac fibroblasts in the pathogenesis of AF and determine if they impact EP maturity by co-culturing them with atrial iPSC-CMs. Specific Aim 3 will elucidate the underlying cellular mechanisms of AF-linked mutations using atrial iPSC-CMs. We will determine the EP phenotype of an SCN5A-E428K and KCNQ1-IAP54-56 mutation using patient-specific atrial iPSC-CMs. In addition, atrial iPSC-CMs from the 2 kindreds will be genetically corrected using CRISPR-Cas9 system to definitively establish causality. Thus, the overarching goals of this proposal are to harness the complementary skills of both Co-PIs (Drs. Darbar and Khetani) to establish the UIC Multi-Ethnic Atrial HiPSC Biorepository to serve as a platform for modeling AF-linked mutations, elucidate the underlying cellular mechanisms, and identify and assess novel mechanism-based therapies for AF. This platform will not only enable a more mechanism-based approach to the treatment of AF but will also `personalize' therapy with improved efficacy and reduced toxicities for individual patients.